Guanine aminohydrolase from rabbit liver has been purified 35,000-fold by a 3 step procedure that makes use of affinity chromatography on 9-(p-aminoethoxyphenyl)guanine Sepharose. Inclusion of thiol in the buffers during purification prevents aggregation. Gel filtration in the presence of thiol gave a value of 110,000 daltons for the molecular weight. Subunit size was shown to be 53,000 daltons by SDS gel electrophoresis in the presence of thiol. These data are consistent with a dimeric structure for native guanine aminohydrolase. The highly purified preparation was shown by polyacrylamide gel electrophoresis to consist of several closely spaced activity bands. Preliminary studies with reagents specific for certain amino acid side chains suggest that cysteine and histidine are necessary for catalytic activity.